The origin of the high stability of 3'-terminal uridine tetrads. The contributions of hydrogen bonding, stacking interactions and steric factors evaluated using modified oligonucleotide analogs.

RNA G-quadruplexes fold almost exclusively into parallel-stranded structures and thus display much less structural diversity than their DNA counterparts. However, also among RNA G-quadruplexes peculiar structural elements can be found which are capable of reshaping physico-chemical properties of the folded structure. A striking example is provided by a uridine tetrad (U-tetrad) placed on the 3'-terminus of a tetramolecular G-quadruplex. In this context the U-tetrad adapts a unique conformation involving chain reversal and is responsible for a tremendous stabilization of the G-quadruplex (ΔTm up to 30 °C). In the current report we attempt to rationalize the origin of this stabilizing effect by concurrent structural, thermal stability and molecular dynamics studies of a series of G-quadruplexes with subtle chemical modifications at their 3'-termini. Our results provide detailed insights into the energetics of the 'reversed' U-tetrad' motif and the requirements for its formation. They point towards the importance of 2'OH to phosphate hydrogen bonding and preferential stacking interactions for the formation propensity and the stability of the motif.